Microalgal feeds containing arachidonic acid and their production and use

ABSTRACT

An animal feed with a high level of arachidonic acid is produced from microalgae, and fed to aquatic animals grown in aquaculture. The arachidonic acid-rich microalgae are fed directly to the aquatic animals, or processed to produce an oil that can be used as a human nutritive supplement. The arachidonic acid-rich microalgae can be combined with long chain omega-3 fatty acids to provide a source of nutrition for humans and animals. The animal feed and nutritional supplements are free of animal byproducts.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to the production and use of certainarachidonic acid (ARA) rich microalgae for or in animal feeds and humanfoods. Such sources of arachidonic acid may be important for theenrichment of feeds used in agriculture, as they are vegetarian innature and a renewable resource. There is a particularly importantapplication in the field of aquaculture, as this environment isparticularly depleted in sources of long chain omega-6 fatty acids, suchas ARA. The algae can be produced in conventional autotrophicproductions schemes or adapted, or engineered, to growth underheterotrophic conditions. The algae can be used as a feed directlythrough pelleting or compounding with other conventional feed sources,or used indirectly as a particulate for the enrichment of zooplankton(e.g., Artemia and rotifers) or other form of bioencapsulation, whichare then consumed by the fish, crustaceans or mollusks. The ARA-richalgal biomass can also be processed to produce a pure triglyceridecontaining the ARA and delivered to humans or animals directly as thecrude or refined oil. Other lipids and byproducts removed during theprocessing of the algal oil as well as the oil-extracted biomass canalso be used as a feed ingredient.

2. Description of Related Art

Many seed oils are highly enriched in the 18-carbon, di-unsaturated,omega-6 fatty acid, linoleic acid (LA), and some even contain the18-carbon, tri-unsaturated omega-6 fatty acid, gamma linolenic acid(GLA). However, terrestrial plants in general do not produce significantquantities of the 20-carbon, tetra-unsaturated, arachidonic acid (ARA).ARA is generally considered a fatty acid of animal origin and is anintegral component of the structure, physiology and general biochemistryof most animals. For example, ARA makes up at least 20% of the fattyacid structure of the lipids of the gray matter of the brain in mammals.Consequently, all herbivorous animals have the ability to convert thedietary LA into ARA through elongation and desaturation enzymes inherentin those species. During periods of an animal's life history, however,preformed dietary ARA may be required due to a particularly highrequirement for the ARA. Such a situation occurs, for example, in humaninfants where there is a period of accelerated brain growth and arequirement for large quantities of this building block of brain tissue.In this case, preformed ARA is delivered from the mother across theplacenta to the fetus in utero and through the breast milk when theinfant is nursing. Thus, sources of ARA are being sought for thesupplementation of infant nutrition products to provide the ARA neededby the growing infant in a form similar to what it would get naturallyfrom its mother.

Although the terrestrial environment is dominated by plants that arerich in omega-6 fatty acids other than ARA, it is deficient in plantsthat produce large amounts of omega-3 fatty acids. Thus, terrestrialanimals, herbivores in particular, tend to be omega-3 deficient. Themarine environment, on the other hand, is dominated by plants(phytoplankton and seaweeds) that are rich in omega-3 fatty acids, butare generally deficient in plants that produce large amounts of omega-6fatty acids. Thus, marine animals, herbivores in particular, canpotentially be omega-6 deficient, and particularly ARA deficient.Heretofore, this fact has not been recognized.

The consequences of omega-3 deficiency in the terrestrial animal diet(particularly man) have been well studied and it is believed that theeffects of many of our chronic illnesses may be relieved in part byincreasing the omega-3 intake in our diet (Le., lowering the ratio ofomega-6 to omega-3 fats in our diet). Hyperimmune responses, plateletaggregation, and vasoconstriction are all thought to be related toexcessive amounts of omega-6 fats in our diet. In an omega-6 deficientdiet, that was replete in omega-3 fatty acids, immunosuppression,increased bleeding times and vasodilatation can be problematic. Sincesuch an omega-6 deficient situation exists in most marine diets, thesupplementation of ARA to such a diet should improve health andresistance to disease. This, in fact, was shown to be the case when anARA-enriched dietary supplement was added to the regular diet of themarine fish Sea bream. In this case, there was an unexpected improvementin resistance to stress and disease when the ARA-supplement was used inthis omega-6 deficient situation. Several products have now been offeredfor sale as aquaculture products containing supplements of ARA. Theseinclude VEVODAR™ (DSM, The Netherlands) and AQUAGROW™ AA (MartekBiosciences Corp, USA).

Both of the above mentioned products are produced using an ARA-rich oilfrom the cultured filamentous fungus Mortierella alpina. The fungus isgrown according to procedures well established in the art and theARA-rich oil is extracted and used for various purposes. In the case ofVEVODAR™, the oil itself is used as a raw material for feedformulations. In the case of AQUAGROW™ AA, the oil is mixed in with afeed matrix and provided as a novel feed formulation. No other sourcesof ARA have been anticipated as being acceptable for feeds or feedapplications to date.

A few microalgae are known to produce ARA, although this is highlyunusual in the marine environment. Such microalgae include, but are notlimited to, Euglenophytes (e.g. Euglena), Rhodophytes (e.g.,Porphyridium), and Chlorophytes (e.g., Parietochlods). Certainmacroalgae (seaweeds) are also known to produce ARA. Such algae, orextracts thereof, would provide a beneficial diet for marine animals aswhole algae, broken algae, or extracted material.

Certain crustaceans (e.g., brine shrimp, Artemia) or other zooplankton(e.g., rotifers) are used as feeds for various aquaculture crops (e.g.,fish or shrimp) as they represent live feed for important first larvalstages. In many cases, the Artemia are “loaded” with certain microalgae,which are carried into the larval animal through the consumption of theArtemia. In this way, certain beneficial nutrients, including ARA, canbe provided from the algae to the larvae through the feed.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide to a composition ofmatter, which is a feed or feed component for animals (vertebrates orinvertebrates), or food or food component for humans, comprising a lipidcontaining arachidonic acid produced from an algal source.

In one embodiment, this invention provides a feed or food comprisingwhole microalgae. The algae can be provided as a wet paste or a drypowder. Whole algal cells can also be processed into pellets by, forexample, standard extrusion processes or tableted using conventionaltableting technology.

In another embodiment of this invention, the ARA-containing microalgaecan be combined with a source of long chain omega-3 fatty acids, such asdocosahexaenoic acid (DHA) or EPA, to provide a balanced nutritivesource of long chain fatty acids. Such DHA or EPA sources may be alsofrom microalgae, from fish or fish byproducts, from marineinvertebrates, invertebrate byproducts or from recombinant organisms(e.g., algae, plants and bacteria).

In another embodimentp of this invention, the microalgae can be brokenin order to facilitate uptake and digestion of the ARA by the animals.Alternatively, the ARA-containing lipids can be extracted and used as asource of ARA alone or in combination with other sources of DHA.

In another embodiment, the ARA-containing feed is free of viruses andinfectious proteins (prions) or nucleic acids. There is great concernover providing animal byproducts to animals as feeds because of thepotential for contamination of the animals with certain viruses orinfectious proteins (prions) that may be associated with animalbyproducts. This invention provides a solution to this problem in thatalgae (microalgae or macroalgae) are not animal sources and are notcarriers of animal viruses, infective proteins, or nucleic acids.

In another embodiment, the ARA-rich feed has enhanced stability. ARA isa highly unsaturated fatty acid and, as such, is very prone tooxidation. The ARA from a fungal sources (e.g. Mortierella alpina) andanimal sources have an inherent oxidative stability problem because ofthis high degree of unsaturation. This oxidation problem is exasperatedby the processing steps required in the preparation of a formulated feedof suitable stability. Polyunsaturated fatty acids, such as ARA from themicroalgal sources, however, have a higher degree of stability than thefungal or animal sources, thereby overcoming the problem of oxidativeinstability.

In another embodiment, the feeds or compositions described above are fedto increase the health or nutritional status of animals or humans.

DETAILED DESCRIPTION OF THE EMBODIMENTS

This invention provides advantages in delivering a stable formulatedfeed that is of vegetable origin, acceptable to the animals, andcontains the correct balance of omega-6 and omega-3 long chainpolyunsaturated fatty acids. Such a feed has not previously been made orcontemplated. Microalgae are already common elements in the aquatic foodchain, so it also represents a natural product for aquatic animals.

Algal cultures including, but not limited to Euglena gracilis,Porphyridium cruentum, Parietochloris incisa and “snow algae” can beobtained from any of a number of publicly accessible culturecollections, university, other public sector groups, or proprietaryindustrial collections. Different algae will produce ARA in variouscomplex lipid forms in different proportions. For example, Porphyridiumcruentum will produce ARA in the form of a phospholipid and glycolipid,while Parietochloris incisa will produce ARA primarily in the form of atriglyceride. The latter case will be preferable because oil-producingalgae, such as Parietochloris, are also more neutrally buoyant in thewater column; thereby providing this species with some unexpectedlybeneficial properties as a neutral buoyancy aquaculture feed.

Edible materials (foods and feeds) can be any materials that areingested and that will not cause harm to the body. One embodiment ofthis invention would be where the algae are genetically modified toproduce excess quantities of ARA, or to produce ARA under heterotrophicgrowth conditions using a reduced carbon source, such as, but notlimited to, glucose, acetate or glutamate. This genetic modification canbe a directed event (ie., the insertion of a specific gene, such as forglucose transport), or it can be a random modification of the alga's owngenome through classical mutagenesis (e.g., ultraviolet light orchemical treatments) then selection of the phenotype of choice.

EXAMPLES

The invention, as contemplated herein, is described in the followingexamples using one ARA-rich alga (Parietochloris) for illustrationpurposes, but it's the invention's utility is not limited to this singleARA-rich alga or to the examples provided.

Example 1

Feed Comprising ARA Microalgal Cell Paste.

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium containing inorganic salts, a nitrogen source such as,but not limited to, urea, nitrate, ammonium ions, phosphate, andpotassium, at a neutral pH. When the culture reaches a biomass densityof at least 5 g/L with an ARA content in excess of 10% by weight of thecells, the culture is harvested by centrifugation or other suitableprocess. The cell paste can be bioencapsulated, for example useddirectly as an enrichment feed for Artemia and rotifers, which are thenprovided as live feed to larval shrimp or fish. Culture volumes can varyfrom 1 Liter flasks in the laboratory, larger scale photobioreactors(e.g., tubular type), 100,000 L tanks, to large paddlewheel-driven algalraceways (several hectares). All procedures are generally the sameexcept for the scale of the process.

Example 2

Feed Composition Comprising ARA Microalgal Cells.

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory, outdoorponds, tanks or photobioreactors. The algae are harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration. The algae are then dried by spray drying, freeze drying,drum drying, vacuum drying, or any other suitable method of drying, toproduce a dry powder containing ARA at a level of at least 10% byweight. This dry powder is then blended with an alternative source ofprotein such as, but not limited to, soy meal, corn meal, fishmeal,casein, pea meal or yeast at a ratio of from one to ten parts algae toone to ten parts protein source. Other binders, such as alginates andflow agents such as diatomaceous earth, can also be added. This driedmaterial can be used directly as a powdered feed containing from 1-20%ARA, or it can be moistened and extruded to make pellets usingconventional extrusion technology. To aid in digestibility, the algaecan be broken prior to drying using common procedures such as, but notlimited to a ball mill, cavitation pressure, or mechanical shearing.Such a feed can be used for larval crustacean culture and fish cultureas well as for animal or human feeds.

Example 3

Feeds Comprising a Blend of ARA-Rich Microalgal Cells and DHA-RichMicroalgal Cells.

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory, outdoorponds, tanks or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration and then dried by spray drying, freeze drying, drumdrying, vacuum drying, or any other suitable method of drying, toproduce a dry powder containing ARA at a level of at least 10% byweight. This dry powder is then blended with an algal powder preparedfrom a DHA-producing alga such as, but not limited to, Crypthecodiniumcohnii. C. cohnii will have a DHA content of at least 10% by weight. Thetwo algal biomasses are mixed in a ratio of from one to ten partsARA-rich algae to one to ten parts DHA-rich algae to provide a DHA/ARAratio from 1:10 to 10:1. Other binders, such as alginates and flowagents such as diatomaceous earth, can also be added. This material canbe used directly as a powdered feed containing from 1-20% ARA or it canbe moistened and extruded to make pellets using conventional extrusiontechnology. Such a feed can be used for larval crustacean or fishculture, animal or human feeds.

Example 4

Feeds Comprising a Blend of ARA-Rich Microalgal Cells and DHA-ContainingMaterials.

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory, outdoorponds, tanks, or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration then dried by spray drying, freeze drying, drum drying,vacuum drying, or any other suitable method of drying, to produce a drypowder containing ARA at a level of at least 10% by weight. ThisARA-rich dry powder is then blended with any DHA-containing materialsuch as, but not limited to, fishmeal, fish oil, squid oil, crustaceanbyproducts, brain meal, or organ meats. The ARA-rich algae andDHA-containing materials are mixed in a ratio to provide a DHA/ARA ratiofrom 1:10 to 10:1. Other binders, such as alginates, and flow agents,such as diatomaceous earth, can also be added. This material can be useddirectly as a powdered feed containing from 1-20% ARA, or it can beextruded to make pellets using conventional extrusion technology. Such afeed can be used for larval crustacean culture and fish culture, as wellas for animal or human feeds.

Example 5

Feeds Comprising ARA Microalgal Lipids.

The ARA-rich microalgae Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory or in outdoorponds, tanks, or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration and then dried by spray drying, freeze drying, drumdrying, vacuum drying, or any other suitable method of drying, toproduce a dry powder containing ARA at a level of at least 10% byweight. The algal lipid is then extracted using nonpolar solvents suchas, but not limited to, hexane, alcohol (isopropanol, ethanol, etc.),supercritical carbon dioxide, or propane. This extract would be called“crude lipid” and will be at least 20% by weight ARA. The crude lipid isthen processed by conventional refining, bleaching and deodorizing toproduce an “edible oil” which will be at least 20% by weight ARA and ispreferably about 30% by weight ARA, more preferably 40% by weight ARAand most preferably 50% by weight ARA or higher. Phospholipids, freefatty acids and other lipid fractions are removed from the crude lipidduring this purification process and are referred to herein as “refiningbyproducts”. Crude ARA lipid, refining byproducts and edible oil can beused in the preparation of animal feeds. The lipid material is blendedwith a source of protein, such as, but not limited to, soy meal, cornmeal, fishmeal, casein, pea meal or yeast, at a ratio of from one to tenparts algal lipid to one to fifty parts protein source. Other binders,such as alginates, and flow agents, such as diatomaceous earth, can alsobe added. This material can be used directly as a powdered feedcontaining from 1-20% ARA, or it can be moistened and extruded to makepellets using conventional extrusion technology. To aid indigestibility, the algae can be broken prior to drying using commonprocedures such as, but not limited to a ball mill, cavitation pressure,or mechanical shearing. Such a feed can be used for larval crustaceanculture or fish culture, as well as animal or human feeds.

Example 6

Feeds Comprising a Blend of ARA Microalgal Lipids and DHA-ContainingMaterials.

The ARA-rich microalga Parietochoris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory, outdoorponds, tanks, or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration then dried by spray drying, freeze drying, drum drying,vacuum drying, or any other suitable method of drying, to produce a drypowder containing ARA at a level of at least 10% by weight. The algallipid is then extracted using nonpolar solvents such as, but not limitedto, hexane, alcohol (isopropanol, ethanol, etc.), supercritical carbondioxide, or propane. This extract would be called “crude lipid”. Thecrude lipid is then processed by conventional refining, bleaching anddeodorizing to produce an “edible oil”. Phospholipids, free fatty acidsand other lipid fractions are removed from the crude lipid during thispurification process and are referred to herein as “refiningbyproducts”. Crude ARA lipid, refining byproducts, and edible oil can beused in the preparation of animal feeds. These lipid products areformulated with DHA-containing materials such as, but not limited to,DHA algae or algal products, fishmeal, fish oil, squid oil, crustaceanbyproducts, brain meal, or organ meats. The algal ARA lipids and DHAmaterials are mixed in a ratio to provide a DHA/ARA ratio from 1:10 to10:1. Other binders, such as alginates, and flow agents, such asdiatomaceous earth, can also be added. This material can be useddirectly as a powdered feed containing from 1-20% ARA, or it can beextruded to make pellets using conventional extrusion technology. Such afeed can be used for larval crustacean or fish culture, animal or humanfeeds.

Example 7

Foods Comprising ARA-Rich Microalgae

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory,outdoorponds, tanks or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration and then dried by spray drying, freeze drying, drumdrying, vacuum drying, or any other suitable method of drying, toproduce a dry powder containing ARA at a level of at least 10% byweight. This dry product can be used directly as a human nutritivesupplement as a powder, colloidal suspension, or tablet.

Example 8

Foods Comprising ARA Microalgal Lipids

The ARA-rich microalga Parietochloris incisa is grown in a fresh waterculture medium as described in Example 1 in the laboratory or in outdoorponds, tanks, or photobioreactors. The culture is harvested bycentrifugation, flocculation, flotation, or any other suitable means ofconcentration and then dried by spray drying, freeze drying, drumdrying, vacuum drying, or any other suitable method of drying, toproduce a dry powder containing ARA at a level of at least 10% byweight. The algal lipid is then extracted using nonpolar solvents suchas, but not limited to, hexane, alcohol (isopropanol, ethanol, etc.),supercritical carbon dioxide, or propane. This extract would be called“crude lipid”. The crude lipid is then processed by conventionalrefining, bleaching and deodorizing to produce an “edible oil”. This oilcan then be added to conventional food matrices including, but notlimited to, infant formulas, toddler formulas, adult formulas, bars, orcereals. The oil can also be packaged in individual dose unitsincluding, but not limited to, soft gelatin capsules, sachets, and foilpouches.

Example 9

Foods Comprising ARA Microalgae and DHA Materials

Foods as described in Example 7 combined with DHA rich materials asdescribed previously to provide a DHA to ARA ratio from 1:10 to 50:1.

Example 10

Foods Comprising ARA Microalgal Lipids and DHA Materials

Foods as described in Example 8 combined with DHA-rich materials asdescribed previously to provide a DHA to ARA ratio from 1:10 to 50:1.

1. A feed for an animal comprising one or more microalga or portionsthereof which comprise arachadonic acid, wherein the microalgalarachadonic acid comprises from about 1% to about 20% of the feed. 2.The feed of claim 1, wherein the arachadonic acid is predominantly in aform chosen from one or more of a phospholipid and a triglyceride. 3.The feed of claim 1, wherein the animal is an aquatic animal.
 4. Thefeed of claim 3, wherein the aquatic animal is aquacultured.
 5. The feedof claim 3, wherein the aquatic animal is a zooplankton.
 6. The feed ofclaim 3, wherein the aquatic animal is chosen from a fish, crustacean,and mollusk.
 7. The feed of claim 5, wherein the zooplankton is chosenfrom a crustacean, brine shrimp, Artemia, and rotifer.
 8. The feed ofclaim 1, wherein the microalga is chosen from one or more of Euglena,Porphyridium, Parietochloris, and snow alga.
 9. A method of delivering afeed comprising arachadonic acid, wherein the arachadonic acid isprovided by a microalga or parts thereof.
 10. The method of claim 9,comprising delivering the feed to an animal.
 11. The method of claim 10,wherein the animal is an aquatic animal.
 12. The method of claim 11,wherein the aquatic animal is aquacultured.
 13. The method of claim 11,wherein the aquatic animal comprises one or more zooplankter.
 14. Themethod of claim 13, wherein the zooplankter is chosen from one or morecrustacean, brine shrimp, Artemia, androtifer.
 15. The method of claim11, wherein the aquatic animal is a fish, crustacean, or mollusk. 16.The method of claim 9, wherein the arachadonic acid is predominantly ina phospholipid form.
 17. The method of claim 9, wherein the arachadonicacid is predominantly in a triglyceride form.
 18. The method of claim 9,wherein the macroalgae are chosen from one or more of Euglene,Porphyridium, Panetochloris, and snow alga.
 19. A microalgal oilcomprising arachadonic acid, wherein the arachadonic acid comprises atleast about 10% (w/v) of the total fat.
 20. The oil of claim 19, whereinthe microalga is chosen from Euglena, Porphyridium, Parietochloris, andsnow alga.
 21. The oil of claim 19, wherein the oil is extracted with asolvent.
 22. The oil of claim 19, wherein the oil further comprisesdocosahexaenoic acid and the ratio of arachadonic acid to docohexaenoicacid ranges from about 1 part arachadonic acid to 10 parts docohexaenoicacid to about 10 parts arachadonic acid to about 1 part docohexaenoicacid.
 23. The oil of claim 22, wherein the source of the docohexaenoicacid is a microalga other than Euglena, Porphyridium, Parietochloris,and snow alga.
 24. The oil of claim 22, wherein the source of thedocohexaenoic acid is fish oil.
 25. The oil of claim 19, wherein thearachadonic acid is predominantly in the form of a phospholipid.
 26. Theoil of claim 19, wherein the arachadonic acid is predominantly in theform of a triglyceride.
 27. A human nutritive supplement comprising oneor more miroalgae or portions thereof, wherein microalgal arachadonicacid comprises at least about 10% by weight of the microalgae.
 28. Thesupplement of claim 27, further comprising a tablet, capsule, colloidalsuspension, or powder.
 29. The supplement of claim 27, furthercomprising a bar; liquid formula for an infant, toddler, or adult; orcereal.
 30. A human nutritive supplement comprising the oil of any ofclaims 19-26.
 31. The supplement of claim 30, further comprising acapsule, colloidal suspension, or liquid formula.
 32. The supplement ofclaim 31, wherein the formula is an infant formula.
 33. The supplementof any of claims 27-32, further comprising docosahexaenoic acid.
 34. Thesupplement of claim 33, wherein the source of the docosahexaenoic acidis one or more microalga.
 35. The supplement of claim 33, wherein thesource of the docosehexaenoic acid is fish oil.
 36. The supplement ofany of claims 33-35, wherein the ratio of docosahexaenoic acid toarachadonic acid ranges from about 1:10 to about 50:1.
 37. Thesupplement of any of claims 27-36, wherein the arachadonic acid ispredominantly in the form of a phospholipid.
 38. The supplement of anyof claims 27-36, wherein the arachadonic acid is predominantly in theform of a triglyceride.